CN107001241A

CN107001241A - Novel ascomycete amides

Info

Publication number: CN107001241A
Application number: CN201580064303.2A
Authority: CN
Inventors: S·鲍曼; J·赫尔曼; K·莫尔; H·斯泰因梅茨; K·格斯; R·拉朱; R·穆勒; R·哈特曼; M·哈米德; W·A·M·埃尔加; M·莫雷诺; F·吉勒; 王亮亮; A·柯书宁; S·忽特尔
Original assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 2014-11-26
Filing date: 2015-11-26
Publication date: 2017-08-01
Anticipated expiration: 2035-11-26
Also published as: CA2968270C; WO2016082934A1; AU2015353077B2; CN107001241B; US10519099B2; JP6654635B2; JP2017537097A; EP3224238A1; AU2015353077A1; US20170327458A1; EP3224238B1; CA2968270A1

Abstract

The present invention provides cystamide of formula (I) and its use for the treatment or prevention of bacterial infections.

Description

Novel ascomycete amides

Cystobacter amides (cystobactimides) are a novel natural product isolated from the myxobacteria cystobacterium atrophaeofaciens (MCy 8071; internal name: cystobacterium atrophaeofaciens).

Cysteamide exhibits good antibacterial activity, particularly against selected gram-negative bacteria such as E.coli, P.aeruginosa and A.baumannii, and has a broad spectrum of activity against gram-positive bacteria.

The invention provides a compound of formula (I)

Wherein,

R¹is hydrogen, OH or is of the formula-O-C_1-6A group represented by an alkyl group;

R²is hydrogen, OH or is of the formula-O-C_1-6A group represented by an alkyl group;

R³is hydrogen, OH or is of the formula-O-C_1-6A group represented by an alkyl group;

R⁴is hydrogen, OH or is of the formula-O-C_1-6A group represented by an alkyl group; and

R⁵is a hydrogen atom or a group represented by the formula:

wherein R is⁶Is OH or NH₂；

Or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutically acceptable formulation thereof.

Expression C_1-6Alkyl refers to a saturated straight or branched chain hydrocarbon group containing 1 to 6 carbon atoms. Expression C_1-4Alkyl refers to a saturated straight or branched chain hydrocarbon group containing 1 to 4 carbon atoms. Examples are methyl (Me), ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

Particularly preferred are compounds of formula (I) wherein:

R¹is hydrogen, OH or is of the formula-O-C_1-4A group represented by an alkyl group;

R²is hydrogen, OH or is of the formula-O-C_1-4A group represented by an alkyl group;

R³is hydrogen, OH or is of the formula-O-C_1-4A group represented by an alkyl group;

R⁴is hydrogen, OH or is of the formula-O-C_1-4A group represented by an alkyl group;and

R⁵is a group of the formula:

wherein R is⁶Is OH or NH₂；

Particularly preferred are compounds of formula (I) wherein R is¹Is OH.

Also preferred are compounds of formula (I) wherein R¹Is of the formula-O-C_1-4A group represented by an alkyl group; in particular wherein R¹Is of the formula-O-CH (CH)₃)₂The groups shown.

Particularly preferred are compounds of formula (I) wherein R is²Is hydrogen.

Particularly preferred are compounds of formula (I) wherein R is²Is OH.

Particularly preferred are compounds of formula (I) wherein R is³Is hydrogen.

Particularly preferred are compounds of formula (I) wherein R is³Is OH.

Particularly preferred are compounds of formula (I) wherein R is³Is of the formula-O-C_1-4A group represented by an alkyl group.

Particularly preferred are compounds of formula (I) wherein R is⁴Is hydrogen.

Particularly preferred are compounds of formula (I) wherein R is⁴Is OH.

Particularly preferred are compounds of formula (I) wherein R is⁵Is a group of the formula:

wherein R is⁶Is OH or NH₂；

Particularly preferred are compounds of formula (II):

wherein R is¹，R²，R³And R⁵A compound of formula (I) as hereinbefore defined, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutically acceptable formulation thereof.

More preferred are compounds of formula (III):

wherein R is²，R³，R⁴And R⁵A compound of formula (I) as hereinbefore defined, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutically acceptable formulation thereof.

Particularly preferred are compounds of formula (IV):

wherein R is¹，R³，R⁴And R⁵A compound of formula (I) as hereinbefore defined, or a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutically acceptable formulation thereof.

Most preferred are the following compounds:

according to a particularly preferred embodiment, the compounds of the invention described herein are at R⁵The group shows the following stereochemical configuration:

wherein R is⁶Is OH or NH₂。

Preferably, the following compounds are excluded from the scope of the present application:

according to a further preferred embodiment, R¹，R²，R³，R⁴And R⁵Not hydrogen at the same time.

Furthermore, preferably, the following compounds are excluded from the scope of the present application:

the invention also provides a pharmaceutical composition comprising one or more compounds of the invention or a pharmaceutically acceptable salt, solvate or hydrate thereof, and optionally one or more carrier substances and/or one or more adjuvants.

The invention also provides the use of said compound or pharmaceutical composition for the treatment and/or prevention of bacterial infections, in particular infections due to escherichia coli, pseudomonas aeruginosa, acinetobacter baumannii or other gram-negative bacteria, as well as gram-positive bacteria.

More preferably, the present invention provides the use of compounds for the treatment and/or prophylaxis of bacterial infections, in particular infections caused by pseudomonas aeruginosa and other gram-negative bacteria.

It is a further object of the present invention to provide a use of a compound or a pharmaceutical composition as described herein for the preparation of a pharmaceutical composition for the treatment and/or prevention of bacterial infections, in particular infections caused by selected gram-negative and gram-positive bacteria.

Examples of pharmacologically acceptable salts of compounds having sufficient basicity are physiologically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid salts; or salts of organic acids such as methanesulfonic acid, p-toluenesulfonic acid, lactic acid, acetic acid, trifluoroacetic acid, citric acid, succinic acid, fumaric acid, maleic acid and salicylic acid. In addition, sufficiently acidic compounds may form alkali or alkaline earth metal salts, such as sodium, potassium, lithium, calcium or magnesium salts; an ammonium salt; or an organic base salt such as methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, meglumine, piperidine, morpholine, tris (2-hydroxyethyl) amine, lysine or arginine salt; all of these are also further examples of salts of the compounds described herein. The compounds of the present invention may be solvates, preferably hydrates. Hydration may occur during the manufacturing process, or as a result of the hygroscopic nature of the initially anhydrous compound. The solvates and/or hydrates thereof may be present, for example, in solid or liquid form.

The respective therapeutic uses of the compounds described herein, their pharmacologically acceptable salts, solvates and hydrates, as well as the formulations and pharmaceutical compositions, are also within the scope of the present invention.

The pharmaceutical composition according to the invention comprises at least one compound according to the invention and optionally one or more carrier substances and/or adjuvants.

As noted above, therapeutically useful agents comprising a compound, solvate, salt or formulation thereof of the present invention are also included within the scope of the present invention. In general, the compounds of the present invention may be administered alone or in combination with any other therapeutic agent by using well known and acceptable means known in the art.

For oral administration, the therapeutically useful and effective agent may be administered by one of the following routes: oral, e.g. as tablets, dragees, coated tablets, pills, semi-solids, soft or hard capsules, e.g. soft or hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral administration including intravenous, intramuscular and subcutaneous injection, e.g. as injectable solutions or suspensions, rectal as a suppository, by inhalation or insufflation, e.g. as a powder preparation such as microcrystals or as a spray (e.g. liquid aerosol), transdermal, e.g. by a Transdermal Delivery System (TDS), e.g. a plaster comprising the active ingredient, or intranasal. To produce such tablets, pills, semi-solids, coated tablets, dragees and hard capsules such as gelatin capsules, the therapeutically useful products may be mixed with pharmaceutically inert, inorganic or organic excipients, such as lactose, sucrose, glucose, gelatin, malt, silica gel, starch or derivatives thereof, talc, stearic acid or its salts, dried skim milk and the like. For the production of soft capsules, excipients, for example, vegetable, petroleum, animal or synthetic oils, waxes, fats and polyols, can be used. For the production of liquid solutions, emulsions or suspensions or syrups, it is possible to use as excipients, for example, water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerol, lipids, phospholipids, cyclodextrins, vegetable oils, petroleum, animal oils or synthetic oils. Particularly preferred are lipids, and more preferred are phospholipids (preferably of natural origin; particularly preferably having a particle size of between 300 and 350 nm), preferably in phosphate buffered saline (pH 7-8, preferably 7.4). For suppositories, excipients which can be used are, for example, vegetable, petroleum, animal or synthetic oils, waxes, fats and polyols. For aerosol formulations, compressed gases suitable for this purpose may be used, for example, oxygen, nitrogen and carbon dioxide. The pharmaceutically useful agents may also contain additives for preservation, stabilization, such as UV stabilizers, emulsifiers, sweeteners, aromatics, salts (for altering the osmotic pressure), buffers, coating additives and antioxidants.

Generally, in the case of oral or parenteral administration to an adult human having a body weight of about 80 kg, a suitable daily dose is about 1mg to about 10,000mg, preferably about 5mg to about 1,000mg, although the upper limit may be exceeded when this is indicated. The daily dose may be a single or divided dose, or for parenteral administration it may be administered by continuous infusion or by subcutaneous injection.

The compounds of the invention can be prepared by fermentation (e.g. by fermentation of the strain MCy8071DSM 27004) or by chemical synthesis application procedures known to the skilled person.

The compounds of the present invention may be synthesized according to the methods described in PCT/EP2014/001925(WO2015/003816), in particular on pages 87 to 138, which are incorporated herein by reference.

For example, the compounds of the present invention can be prepared by the following scheme:

examples

1. Fermentation of

Preparation conditions

Production strain

The strain, deep brown cyst bacillus (Cystobacter velatus) MCy8071 belongs to the order myxobacteria, the sub-order cystobacteriaceae, the family cystobacteriaceae, the genus Cystobacter. A comparison of part of the 16S rRNA gene sequence with the sequences of public databases (BLAST, a "basic local alignment search tool" provided by NCBI (national center for biotechnology information) showed 100% similarity to the deep brown cyst bacillus strain DSM 14718.

Among the Chinese soil samples collected in 1982, MCy8071 was isolated at the Helmholtz infection research center (HZI, formerly GBF). The strain was deposited at the German Collection of microorganisms (DSM) of Brenrick (Braunschweig) at 3 months 2013 under the number DSM 27004.

Culturing

Strain MCy8071 on Yeast agar (VY/2: 0.5% Saccharomyces cerevisiae, 0.14% CaCl)₂×2H₂O, 0.5. mu.g vitamin B12/l, 1.5% agar, pH7.4), CY-agar (tyrosone 0.3%, yeast extract 0.1%, CaCl₂×2H₂O0.1%, agar 1.5%, pH7.2) and P-agar (macocone peptone (peptone Marcor) 0.2%, starch 0.8%, single cell protein prodione 0.4%, yeast extract 0.2%, CaCl₂×2H₂O 0.1％，MgSO₄0.1%, Fe-EDTA8mg/l, 1.5% agar, pH 7.5). Working cultures were incubated in liquid medium CY/H (50% CY medium +50mM Hepes buffer, 50% H medium: 0.2% soy flour, 0.8% glucose, 0.2% starch, 0.2% yeast extract, CaCl₂×2H₂O)0.1％，MgSO₄0.1%, Fe-EDTA8mg/l, Hepes buffer 50mM pH 7.4).

The liquid culture was shaken at 180rpm at 30 ℃. For storage, 2ml portions of three-day-old cultures were stored at-80 ℃. There were no problems with reactivation even over the years on the agar plates described above or in 20ml of CY/H medium (in 100ml Erlenmeyer flasks with stopcocks and aluminium caps). After one or two days, 20ml of the culture may be scaled up to 100 ml.

Description of the morphology

The rod-shaped cells of the above-mentioned strain MCy8071 had a length of 9.0 to 14.5 μm and a width of 0.8 to 1.0 μm after two days in the liquid medium CY/H. On the above agar plate, colonies were circular. In VY/2-agar, colonies are thin and transparent. In VY/2-agar, yeast degeneration is visible. In CY-agar, the culture appeared transparent orange. On P-agar, the generation of cell populations is unique and colony aggregation behavior is reduced. Colonies were orange-brown. The starch in the P-agar is broken down.

MCy8071 is resistant to the following antibiotics: ampicillin, gentamicin, hygromycin, polymyxin, bacitracin, spectinomycin, neomycin and fusidic acid. In the presence of cephalosporins and kasugamycinThere was a possibility of weak growth, and growth in the presence of thiostrepton, trimethoprim, kanamycin, and oxytetracycline was not possible (the final concentration of all antibiotics was adjusted to 50. mu.g ml^-1)。

Production of ascomycete amides

The strain is produced in a complex medium. It prefers nitrogen-containing nutrients such as single cell protein (Probion) and protein breakdown products such as peptone, tryptone, yeast extract, soybean meal and meat extract. In this case, the production in the presence of a plurality of the protein mixtures is due to the use of a single protein.

Cystamide is produced between the logarithmic phase to the stationary phase relative to the growth stationary phase. After two days in 100 l fermentation (medium E), the amount of product did not increase any more.

Cystamide is delivered to the medium and bound to XAD-adsorbent resin. XAD was sieved through a metal sieve and eluted in acetone. Tests were carried out at different production temperatures (21 ℃, 30 ℃, 37 ℃ and 42 ℃) and were not able to be carried out at 42 ℃. The optimum temperature at maximum ventilation is 30 ℃.

MCy8071 was fermented using 100 liters of medium E (skim milk 0.4%, soy flour 0.4%, yeast extract 0.2%, starch 1.0%, MgSO 4₄0.1 percent, 8mg/l of Fe-EDTA and 0.5 percent of glycerol; pH7.4), and with 70 l of medium M (Soytone 1.0%, maltose 1.0%, CaCl₂×2H₂O 0.1％，MgSO₄0.1 percent and 8mg/l of Fe-EDTA; pH7.2), in a 100 l fermenter and fermented at 30 ℃ for 4 days. The pH was adjusted with potassium hydroxide (2.5%) and sulfuric acid between 7.2 and 7.4. The stirrer speed was 100-. The dissolved oxygen content in the fermentation liquor is adjusted to pO from the rotating speed of the stirrer₂40 percent. 1% adsorbent resin was added to the fermentation broth to bind cystamide. 5 l of 3-day-old preculture (E or M medium, respectively) were inoculated into the fermenter. Analysis by HPLC-MS and methanol extraction against E.coliSerial dilution experiments of the extract checked for product during fermentation. The strain produces ascomycylamine.

The following cystamide (except cystamide a, B, C, D, E and F described in WO2015/003816 ═ PCT/EP 2014/001925) have been isolated and characterized by NMR and MS:

ascomycete amide 935-2:

MS:

NMR:

cystosporanide 935-2 NMR (700MHz, MeOH-d)₄)

Ascomycete amide 819-1:

cystamide 845-2:

ascomycetes amide 846-1:

ascomycoamide 861-1:

ascomycete amide 862-1:

ascomycete amide 862-2:

ascomycete amide 891-1:

ascomycete amide 903-1:

ascomycete amide 903-2:

ascomycete amide 905-1:

ascomycete amide 905-2:

ascomycete amide 920-1:

ascomycete amide 933-1:

ascomycete amide 933-2:

ascomycete amide 934-1:

ascomycete amide 934-2:

sphaerotheca amide 919-2:

C₄₆H₄₆N₇O₁₄[M+H]⁺HRMS (ESI) of (4) calculated value 920.3103, detected value 920.3106.

Ascomycete amide 919-2 in MeOH-d₄Nuclear magnetic data of (1):

ascomycete amide 919-2 in DMSO-d₆Nuclear magnetic data of (1):

signals corresponding to these units cannot be assigned due to signal broadening effects in NMR spectra in DMSO-d 6: see also "structural elucidation" and FIGS. S42-S45.

Cysteamine amide containing a methoxy-asparagine (or aspartic acid) fragment as in the normal peptide shows the 413(414) -fragment in its mass spectrum (figure 1). When methoxy-aspartic acid (aspartic acid) is present, the iso-amino acid-containing cystamide does not display the 413(414) -fragment (fig. 2). The presence of iso-and non-iso amino acids can be elucidated based on the presence of this fragment in the mass spectrum of cystamide.

Biological evaluation of cystamide

Antibacterial activity

Cystosporanamides (Cys)919-2,920-1,934-2,935-2,891-2 and 905-2 were evaluated together with the already described derivatives (861-2,877-2,920-2) and were used against a selected group of gram-negative bacteria. The derivatives 861-2,877-2,919-1 and 920-2 correspond to cysteamine F, H, A and B described in WO 2015/003816. MIC values are expressed in μ g/ml; ciprofloxacin (CP) was used as reference.

Cystamide 919-2 and 891-2 were tested together with the already described derivatives 861-2(F) and 919-1(A) on a larger microbiome and CHO-K1 cell line.

MIC values are expressed in μ g/ml;^*IC50 in μ M; use of Ciprofloxacin (CP) as reference

Drug resistance rate

The resistance to cystamide 861-2 and 919-2 was determined using E.coli DSM-1116 at 4-fold MIC and was 10^-7-10^-8。

In vitro Activity

The activity of cystamide 861-2 on E.coli and P.aeruginosa gyrase DNA supercoiled (sc) activity was determined in comparison to cystamide 919-2 and Ciprofloxacin (CP).

Genotoxicity

No detectable genotoxic effect was observed in the micronucleus formation assay of the CHO-K1 cell line using 20. mu.g/ml cystamide 861-2,919-2 and ciprofloxacin. Mitomycin C (100ng/ml) was used as a positive control. All experiments were performed in triplicate and microscopic images of stained nuclei were evaluated. Micronuclei formation was clearly observed in mitomycin C treated CHO-K1 cells, but not in untreated control, ciprofloxacin and cystamide treated cells.

Materials and methods

Minimum Inhibitory Concentration (MIC) determination

The indicator strains of bacteria used in the susceptibility tests are part of our strain collection, either from the German Collection of microorganisms and cell cultures (DSMZ) or from the American Type Culture Collection (ATCC). Wild Type (WT) E.coli strains and E.coli mutants were professor P.Heisig, doctor Hui gift, of medicinal biology and microbiology, Hamburg university. Coli strains JW0401-1(WT) and. DELTA.tsx were obtained from the collection of CGSCs.

MIC values were determined by microdilution assay. Overnight cultures were grown appropriatelyDilution in Medium to obtain 10⁴-10⁶cfu/mL inoculum. Yeast in Myc Medium (1% phytone, 1% glucose, 50mM HEPES buffer, pH7.0), Streptococcus pneumoniae and enterococcus in tryptone Soy Medium (TSB: 1.7% peptone casein, 0.3% peptone soy meal, 0.25% glucose, 0.5% NaCl, 0.25% K)₂HPO₄pH 7.3), M.smegmatis grown in Middlebrook (Middlebrook)7H9 medium supplemented with 10% Middlebrook ADC enrichment and 2 ml/l glycerol all other listed bacteria were grown in Miller-Citon (M ü ller-Hinton) medium (0.2% beef infusion solids, 1.75% casein hydrolysate, 0.15% starch, pH7.4), cystamide and control drugs were added directly to the culture in sterile 96-well plates in duplicate and serial dilutions were prepared, microorganisms were grown on a microplate (750rpm, 30-37 ℃, 18-48 hours) except under non-shaking conditions (37 ℃, 5% CO)₂18 hours). Growth inhibition was assessed by visual inspection and MIC was defined as the lowest concentration of compound that inhibited visible growth.

Cytotoxicity

CHO-K1 cells were obtained from DSMZ and cultured under conditions recommended by the depositor.cells were plated at 6 × 10³Individual cells/well were seeded in 180 μ l complete medium in 96-well plates and treated with serial dilutions of compounds after 2 hours of equilibration. Each sample was tested in duplicate and an internal DMSO control. After 5 days of incubation, 20. mu.l of 5mg/ml MTT (thiazole blue tetrazolium bromide) in PBS was added to each well and incubated at 37 ℃ for a further 2 hours. The medium was then discarded, and the cells were washed with 100. mu.l PBS, followed by addition of 100. mu.l of 2-propanol/10N HCl (250:1) to dissolve formazan particles. Absorbance at 570nm was measured using a plate reader (tecanifinal M200Pro) and expressed as a percentage of cell viability relative to the corresponding methanol control.

Drug resistance rate

To determine the spontaneous resistance to cystamide, in Miller-Citon (M ü l)ler-Hinton) medium to a final concentration of 10¹⁰CFU/mL, and different volumes were scored on duplicate agar plates containing cystamide at 4-fold MIC on E.coli. In addition, several dilutions of E.coli cultures were streaked on antibiotic-free plates. After 1 day, the resistance rate was determined by dividing the CFU on cystamide containing plates by the number of CFU on antibiotic free plates.

Enzyme inhibition

To test the gyrase activity of ascomyces amides, commercially available E.coli and P.aeruginosa gyrase supercoiled kits (Inspiralis, Novie, UK) were used. For standard reactions, 0.5 μ g of relaxed plasmid was mixed with 1 unit of gyrase in 1 × reaction buffer (see kit manual) and incubated at 37 ℃ for 30 minutes. The reaction was quenched by addition of DNA gel loading buffer containing 10% (w/v) SDS. Samples were separated on a 1% (w/v) agarose gel and the DNA was visualized with EtBr. All stock solutions and dilutions of the natural products were prepared in 100% DMSO and added to the supercoiled reaction to give a final DMSO concentration of 2% (v/v).

Genotoxicity study

Chinese hamster ovary CHO-K1 cells (ACC-110) were obtained from DSMZ and stored under conditions recommended by the depositor for genotoxicity studies, cells were plated at 5 × 10³Cells/well were seeded in black 96-well plates with an optical bottom and allowed to adhere for 1 day before compound addition. CP, cystamide and mitomycin C were added to final concentrations of 20. mu.g/ml (gyrase inhibitor) and 100ng/ml (mitomycin C). Cells were treated for 48 hours, washed twice with phosphate buffered saline (PBS, pH7.4) and fixed with AcO/MeOH (1:1, -20 ℃) for 10 minutes at room temperature. After repeated washing with PBS, cells were stained with 5. mu.g/mL Hoechst33342 in PBS for 15 minutes under protection from light at room temperature. After washing, the samples were imaged (200 x magnification) on an automated microscope (Pathway855, BD bioscience) with a filter suitable for Hoechst. All samples were prepared and analyzed in two separate experimentsMicronucleus formation was analyzed in triplicate in the assay.

3. Synthesis of cystobacter amide C derivatives

3.1 Synthesis of the Individual rings used in the variants

The preparation of different monocycles for use during the synthesis of cystamide C derivatives is described herein.

Preparation of the C Ring

Preparation of ring B

3.2 coupling of the B and C rings to give different prepared BC fragments

Coupling of the a-ring with the BC-fragment (BC1, BC2, BC3) to synthesize ascocarboxamide C derivatives (1s) - (3 s).

Compound (I)	R₁	R₂
			(1s)	OH	OMe
(2s)	OiPr	OiPr
			(3s)	OMe	OH

3.4 preparation of Compound 4s

3.5. Test of

3.5.1. General experimental information

Starting materials and solvents were purchased commercially from commercial suppliers and used without further purification. All chemical yields refer to the purified compound, without optical purification. The reaction process was carried out by TLC silica gel 60F₂₅₄Aluminum sheets were monitored and visualized by 254nm UV. Using silica gel(40-63 μm) was performed in a waters instruments containing 2767 sample manager, 2545 binary gradient module, 2998PDA detector and 3100 electrospray mass spectrometer using waters XBridge column (C18,150 × 19mm, 5 μm), binary solvent system a and B (a ═ water with 0.1% formic acid; B ═ MeCN with 0.1% formic acid) as eluent, purified at a flow rate of 20mL/min, 8min with a gradient of 60% -95% B, melting point was measured on a Stuart Scientific melting point instrument 3 (bibbysterin, uk), uncorrected, at 300K, on a bruke DRX-500(1H, 500 MHz; 13C, 126MHz) or bruke fourier 300(1H, 300 MHz; 13C, 75MHz) spectrometer, NMR spectra were recorded as shifts, as internal standard for hydrogenation of residues in CDCl (CDCl), reference solvent residue (CDCl)₃：＝7.26,77.02；DMSO-d₆2.50, 39.99. the fragmentation pattern is described as apparent multiplicities and is designated as s (singlet), br s (broad singlet), D (doublet), dd (doublet), t (triplet), q (quartet), m (multiplet). coupling constants (J) are given in hertz (Hz). LC/MS Finnigan Surveyor MSQ Plus (seemer fly technologies, delaische, germany) the purity of all compounds used in the bioassay is 95% or more, the system consists of LC pumps, auto-samplers, PDA detectors and single quadrupole MS detectors, and a standard software Xcalibur for operation. a chromatography column (Macherey-Nagel GmbH, D ü hren, D) is performed as a stationary phase using RP C18 nuclodur 100-5(125 × 3mm) a binary solvent system a and B (a. with 0.1% of a. the flow rate of 0.1% of water; 0.1% of TFA) is set as a flow rate of 0.10V at a flow rate of 0.10 min and a flow rate of water is maintained at a flow rate of 0.10% of 0.10 min under an initial gradient of 10V at 10 minPositive mode and uv tracking spectrum obtained at 254 nm.

3.5.2. General synthetic procedure:

a) a solution of the acid (25mmol), isopropyl bromide (52mmol) and potassium carbonate (52mmol) in 100ml DMF was heated at 90 ℃ overnight. Excess DMF was removed under reduced pressure and the remaining residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and then excess solvent was removed under reduced pressure to give the pure product.

b) To a solution of the nitro derivative (10mmol) in EtOH (60mL) at 55 deg.C under stirring was added iron powder (2.80g,50mmol) and NH₄A solution of Cl (266mg,5mmol) in water (30 mL). The reaction mixture was refluxed for 1-2 hours, then the iron was filtered while hot and the filtrate was concentrated to dryness in vacuo. The residue was diluted with water (30mL) and NaHCO₃Basified (saturated aqueous solution) to pH 7-8. The mixture was extracted with EtOAc. The combined organic phases were washed with brine and dried (MgSO)₄) The solvent was removed by evaporation in vacuo. The resulting crude material was triturated with n-hexane and collected by filtration.

f) To a solution of aldehyde (4mmol) and NaOH (0.8g,20mmol) in water (50mL) was added AgNO portionwise with stirring₃(3.4g,20 mmol). The reaction was refluxed overnight, then cooled and filtered through celite. The filtrate was cooled in an ice bath and acidified to pH 3-4 with 37% HCl. The precipitated solid was collected by filtration, washed with cold water, then n-hexane.

h) To an acid (2mmol), an amine (2.4mmol) in anhydrous CHCl under nitrogen with stirring₃To the solution (50mL) was added dichlorotriphenylphosphine (3.0g,9 mmol). The reaction was heated at 80 ℃ for 5 h. The solvent was removed by vacuum distillation. The residue was purified by flash chromatography.

i) Amination according to procedure 2 reported below¹. Boiling a solution of acid (1mmol) and amine (1mmol) in 2.5ml of xylene with 2M PCl₃CH (A) of₂Cl₂(0.4mmol) was treated. After 2h, excess solvent was removed by evaporation and the residue was purified by column chromatography.

j) Hydrolysis was performed according to procedure 1 reported below². Ester (0.1mmol), sodium hydroxide 1M (3 mL)) And anhydrous methanol at 45 ℃ overnight, cooling, the reaction mixture was acidified to pH 1(3mL, 1M hydrochloric acid) and extracted with dichloromethane (3 × 150mL), the organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to give the pure product.

3.5.3. Special synthesis procedure:

2-formyl-6-methoxyphenyl acetate

To a solution of 3-methoxysalicylaldehyde (4.56g, 30mmol) and pyridine (2.43mL, 30mmol) in DCM (40mL) was added acetyl chloride (2.36g, 30mmol) dropwise with stirring. The reaction was stirred at room temperature overnight, and then the solvent was removed by vacuum distillation. The residue was diluted in cold dilute hydrochloric acid. Filtered and washed with cold water and then n-hexane. Yield 94% (off-white solid), M/z (ESI +)195[ M + H]⁺。

6-formyl-2-methoxy-3-nitrophenylacetic acid

To ice-cooled 2-formyl-6-methoxyphenyl acetate (1.94g, 10mmol) and KNO with stirring₃(1.01g,10mmol) in CHCl₃Trifluoroacetic anhydride (12mL) was added to the suspension (15 mL). The reaction was stirred in an ice bath for 2h, then at room temperature overnight. The reaction was very carefully diluted with water (50mL) and CHCl₃And (4) extracting. The combined organic extracts were dried (MgSO)₄) The solvent was removed by evaporation in vacuo. The residue was dissolved in toluene and purified using flash chromatography (SiO)₂n-hexane-EtOAc ═ 3: 1). Yield 45% (yellow semisolid), M/z (ESI +)239[ M [)]⁺。

2-hydroxy-3-methoxy-4-nitrobenzaldehyde

To a suspension of 6-formyl-2-methoxy-3-nitrophenylacetic acid (957mg,4mmol) in water (30mL) was added NaOH (0.8g,20mmol) with stirring. The reaction was refluxed for 2h and then stirred at room temperature overnight. The solution was cooled in an ice bath and acidified to pH 3-4 by 2M HCl. The precipitated solid was collected by filtration, washed with cold water, then n-hexane. Yield 90% (yellow brown solid), M/z (ESI +)197[ M [)]⁺。

2, 3-dihydroxy-4-nitrobenzaldehyde

To a stirred solution of 18(1.2g, 5mmol) in DCM (10mL) cooled at 0 ℃ in an ice bath was added BBr carefully under a nitrogen atmosphere₃(1M in DCM, 20 mL.) the reaction mixture was warmed to room temperature and further stirred overnight, the solvent was removed in vacuo, the residue was carefully diluted with water (50mL), if necessary, the medium was acidified to pH 4-5 with 2N HCl, the mixture was extracted with EtOAc (3 × 30mL), the combined organic extracts were washed with brine, anhydrous MgSO₄Dried and the solvent removed by vacuum distillation. The residue was dissolved in CHCl₃And using flash chromatography (SiO)₂DCM-MeOH ═ 98:2) purification.

3.5.4. Derivative test data (1s-4s)

4- (4- (4-aminobenzoylamino) -2-hydroxy-3-methoxybenzoylamino) -3-isopropoxybenzoic acid (1s)

The yield is 85%; light yellow crystals;¹H NMR(500MHz,DMSO-d₆)12.79(br s,1H),11.38(br s,1H),10.98(br s,1H),9.22(br s,1H),8.56(d,J＝8.5Hz,1H),7.80(d,J＝8.8Hz,1H),7.73(d,J＝8.5Hz,2H),7.65(d,J＝8.8Hz,1H),7.59(dd,J＝8.5,1.6Hz,1H),7.57(d,J＝1.6Hz,1H),6.69(d,J＝8.5Hz,2H),5.39(br s,2H),4.76(septet,J＝6.0Hz,1H),3.78(s,3H),1.39(d,J＝6.0Hz,6H)；¹³C NMR(126MHz,DMSO-d₆)166.99,165.03,163.28,151.46,149.53,146.13,139.38,136.34,133.45,129.43,125.62,125.55,122.65,121.21,119.28,115.71,113.89,113.75,113.43,71.72,60.40,21.73；m/z(ESI+)479.99[M+H]⁺；t_R＝14.53min。

4- (4- (4-aminobenzoylamino) -2, 3-diisopropoxybenzoylamino) -3-isopropoxybenzoic acid (2s)

The yield is 81%; a beige solid;¹H NMR(500MHz,DMSO-d₆)12.82(br s,1H),10.36(br s,1H),9.06(br s,1H),8.60(d,J＝8.5Hz,1H),8.01(d,J＝8.8Hz,1H),7.75(d,J＝8.8Hz,1H),7.70(d,J＝8.8Hz,2H),7.61(dd,J＝8.5,1.9Hz,1H),7.58(d,J＝1.9Hz,1H),6.63(d,J＝8.8Hz,2H),5.90(br s,2H),4.75(septet,J＝6.0Hz,1H),4.63(septet,J＝6.3Hz,1H),4.52(septet,J＝6.0Hz,1H),1.35(d,J＝6.0Hz,6H),1.31(d,J＝6.0Hz,6H),1.27(d,J＝6.3Hz,6H)；¹³C NMR(126MHz,DMSO-d₆)166.88,164.45,162.79,152.66,148.60,145.71,141.15,137.69,132.89,129.08,125.58,125.44,123.52,122.83,119.87,118.64,117.50,113.94,112.87,77.12,75.70,72.02,22.25,21.90,21.79；m/z(ESI+)549.86[M+H]⁺；t_R＝13.10min。

4- (4- (4-aminobenzoylamino) -3-hydroxy-2-methoxybenzoylamino) -3-isopropoxybenzoic acid (3s)

The yield is 79%; a beige solid;¹H NMR(500MHz,DMSO-d₆)12.67(br s,1H),10.90(s,1H),10.12(s,1H),9.73(s,1H),8.65(d,J＝8.4Hz,1H),7.80–7.71(m,2H),7.64–7.54(m,4H),6.67–6.59(m,2H),5.95(br s,2H),4.86(septet,J＝6.2Hz,1H),3.99(s,3H),1.41(d,J＝6.0Hz,6H)；13C NMR(126MHz,DMSO-d₆)166.97,166.24,162.25,152.99,147.98,145.57,141.60,133.01,132.60,129.79,125.45,122.58,121.24,119.02,118.71,118.20,112.99,112.96,112.69,71.00,61.60,21.71.m/z(ESI+)480.08[M+H]⁺；tR＝10.70min。

4- (4- (4-aminobenzoylamino) -3-isopropoxy-2-methoxybenzoylamino) -3-isopropoxybenzoic acid (4s)

The yield is 43%; a beige solid;¹H NMR(500MHz,DMSO-d₆)12.82(br s,1H),10.90(br s,1H),9.09(br s,1H),8.62(d,J＝8.2Hz,1H),8.06(d,J＝8.8Hz,1H),7.84(d,J＝8.8Hz,1H),7.70(d,J＝8.5Hz,2H),7.60(dd,J＝8.2,1.6Hz,1H),7.58(d,J＝1.6Hz,1H),6.63(d,J＝8.5Hz,2H),5.92(br s,2H),4.85(septet,J＝6.0Hz,1H),4.47(septet,J＝6.0Hz,1H),4.04(s,3H),1.40(d,J＝6.0Hz,6H),1.32(d,J＝6.0Hz,6H)；¹³C NMR(126MHz,DMSO-d₆)166.96,164.45,161.87,152.74,151.59,145.55,140.72,138.04,133.03,129.11,125.79,125.47,122.67,120.61,119.78,118.58,117.31,113.14,112.87,76.50,71.14,61.78,22.36,21.66；m/z(ESI+)522.04[M+H]⁺；t_R＝15.58min。

reference documents:

1) salicylanilide inhibitors of Alina fosvska, Richard d.wood, Ernest Mui, jitnter p.dubey, leandrar.ferreira, Mark r.hickman, Patricia j.lee, Susan e.leed, Jennifer m.auchwitz, William j.welsh, Caroline Sommerville, Stuart Woods, Craig Roberts and Rima mcleod. Journal of medicinal chemistry (salicyliilide Inhibitors of toxoplasmographical ondii.j. med. chem.), 2012,55(19), pp 8375-.

2) Valeria Azzarito, Pancami Prahakaran, Alice I.Bartlett, Natasha Murphy, Michael J.Hardie, Colin A.Kilner, Thomas A.Edwards, Stuart L.Werriner, Andrew J.Wilson.2-O-alkylated p-benzamide alpha-helix mimetics: the effect of stent curvature. Organic and biomolecular chemistry (2-O-Alkylated Para-Benzamide alpha-Helix Chemicals: The Role of ScaffoldCurvature. org. biomol. chem.), 2012,10,66469.

Claims

1. A compound of formula (I)

Wherein,

R³is hydrogen, OH or is of formula-O-C_1-6A group represented by an alkyl group;

R⁵is a hydrogen atom, or a group represented by the formula:

or

Wherein R is⁶Is OH or NH₂；

2. The compound of claim 1, wherein

R⁴is hydrogen, OH or is of the formula-O-C_1-4A group represented by an alkyl group; and

R⁵is a group of the formula:

or

Wherein R is⁶Is OH or NH₂；

3. The compound of claim 1 or 2, wherein R¹Is OH.

4. The compound of claim 1 or 2, wherein R¹Is of the formula-O-C_1-4A group represented by an alkyl group; in particular wherein R¹Is of the formula-O-CH (CH)₃)₂The groups shown.

5. A compound according to any one of the preceding claims 1 to 4, wherein R is²Is hydrogen.

6. A compound according to any one of the preceding claims 1 to 4, wherein R is²Is OH.

7. A compound according to any one of the preceding claims 1 to 6, wherein R³Is hydrogen.

8. A compound according to any one of the preceding claims 1 to 6, wherein R³Is OH.

9. A compound according to any one of the preceding claims 1 to 6, wherein R³Is of the formula-O-C_1-4A group represented by an alkyl group.

10. A compound according to any one of claims 1 to 9, wherein R is⁴Is hydrogen.

11. A compound according to any one of claims 1 to 9, wherein R is⁴Is OH.

12. A compound according to any one of claims 1 to 11, wherein R is⁵Is a group of the formula:

or

Wherein R is⁶Is OH or NH₂。

13. A compound selected from the group consisting of:

14. a compound selected from the group consisting of:

15. a pharmaceutical composition comprising a compound according to any preceding claim, and optionally one or more carrier substances and/or one or more adjuvants.

16. Use of a compound or pharmaceutical composition according to any preceding claim for the treatment or prevention of a bacterial infection.